Project Summary/Abstract The epidermal growth factor receptor (EGFR) is a ubiquitously expressed cell-surface tyrosine kinase that regulates cell survival, proliferation, and differentiation through the activation of downstream pathways including the PI3K/Akt and MAPK cascades. Loss of EGFR is embryonic lethal and dysregulation of EGFR is linked to the progression of many forms of cancers. This has made EGFR an intense area of study and has led to several FDA approved therapeutics, including small molecule tyrosine kinase inhibitors (e.g. gefitinib, erlotinib) and inactivating antibodies (e.g. cetuximab, panitumumab). EGFR is thought to initiate signaling through the MAPK and PI3K/Akt pathways by first dimerizing, then autophosphorylating and recruiting the initiating signaling adapter proteins. This process is opposed by spatial reorganization of the receptors to clathrin-coated pits, where their internalization and intracellular trafficking blocks additional signaling. However, several studies are beginning to challenge this simple view of EGFR signaling, suggesting that spatial reorganization of phosphorylated EGFR may activate, as well as antagonize, downstream signaling on different timescales. Unfortunately, strong experimental evidence in support of this conjecture has been lacking due to the inability to decouple receptor phosphorylation from spatial reorganization during ligand-mediated activation. I hypothesize that the spatial reorganization of phosphorylated EGFR (phosphoEGFR) to oligomeric clusters is necessary for the activation of MAPK signaling when full-length receptor is expressed at physiologically relevant levels. To test this hypothesis, we have developed chemical tools that allow for stimulation of complete receptor phosphorylation independent from spatial reorganization. Using a chemically dimerizable, full-length EGFR (cdEGFR), our approach activates the tyrosine kinase activity of full-length EGFR, which is sufficient to recruit key adaptor proteins such as Grb2 and SOS. However, these activated receptors do not reorganize on the cell surface or potentiate signaling through MAPK or Akt. I will test several hypotheses to identify the mechanism by which spatial reorganization of EGFR stimulates signal transduction through downstream pathways.